Cell Junction

The cell junctions of the portion of the capillary walls that are in contact with alveoli are tight, maintaining the relative "dryness" of the alveoli by limiting fluid movement from the capillaries to the alveoli.

From: Clinical Imaging (Third Edition) , 2014

Intercellular Junctions

In Cell Biology (Third Edition), 2017

Investigation of junctions began when microscopists and physiologists recognized that epithelial and muscle cells adhere to each other and the underlying extracellular matrix. They also discovered that some epithelia form a tight barrier between the luminal surface and the underlying tissue spaces. The physical basis of these interactions became clear during the 1960s, when electron micrographs of thin sections of vertebrate tissues revealed four types of intercellular junctions that connect the plasma membranes of adjacent cells (Table 31.1 and Fig. 31.1) and two types of junctions to bind to the extracellular matrix. Subsequent research established the molecular architecture of these junctions, each based on a different transmembrane protein:

Adherens junctions: Transmembrane proteins called cadherins (see Fig. 30.5) link neighboring cells and connect to actin filaments in the cytoplasm.

Desmosomes: Another type of cadherin links cells together and connects to cytoplasmic intermediate filaments.

Tight junctions: Transmembrane proteins called claudins join the plasma membranes of two cells to create a barrier that limits diffusion of ions and solutes between the cells and molecules between apical and basolateral domains of the plasma membrane.

Gap junctions: Transmembrane proteins called con­nexins form channels for small molecules to move between the cytoplasms of neighboring cells.

Hemidesmosomes: Integrins (see Fig. 30.9) connect cytoplasmic intermediate filaments to the basal lamina across the plasma membrane.

Focal adhesions: Integrins associated with cytoplasmic actin filaments adhere to the extracellular matrix.

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Gametogenesis, Fertilization and Early Development

Barry R. Zirkin , Erwin Goldberg , in Encyclopedia of Reproduction (Second Edition), 2018

Intercellular Bridges

Intercellular bridges connect all the spermatids originating from the same stem cell, a consequence of the incomplete cytokinesis of spermatogonia during mitosis and of spermatocytes during meiosis. Disruption of the intercellular bridges can cause male infertility. The importance of intercellular bridges is clear. Thus, movement of molecules among haploid cells is made possible by intercellular bridges, and this movement is likely to be involved in the sharing of mRNAs among germ cells. This sharing, in turn, is important for sperm production because numerous essential proteins are encoded on sex chromosomes (Kim et al., 2015).

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Epithelial Tissues

Donald B. McMillan , Richard J. Harris , in An Atlas of Comparative Vertebrate Histology, 2018

Desmosomes and Intercellular Bridges

Intercellular connections occur in many epithelia but they are most conspicuous in the lower layers of stratified epithelia, especially those subjected to mechanical stress. Note the strands between the cells of the deeply stained "prickle cells" in sections of mammalian epidermis (Fig. C55). Processes or "prickles" from adjacent cells meet and are firmly attached to each other by a desmosome, which stains as a dense dot. Because of shrinkage during preparation of the tissue, the cells pull away from one another while the desmosomes hold, thereby producing a spiny appearance. (Visualize buttons straining on a fat man's shirt.) The intercellular connections, which have been artificially stretched, have long been known as intercellular bridges although there is no cytoplasmic continuity between adjacent cells.

Figure C55. Photomicrograph of a section of human skin showing "prickle cells" in the stratified squamous epithelium. The cells are held together by attachment plaques, the desmosomes, that hold when the cells shrink during fixation. intercellular bridges between adjacent cells are an artifact produced by shrinkage of the cells; there is no cytoplasmic continuity between adjacent cells. 63×.

Electron micrographs show that desmosomes consist of two dense attachment plaques fused with the inner leaflet of the cell membrane of adjacent cells, so that each cell contributes one half of the desmosome (Figs. C56 and C57). Each half desmosome is anchored to its cell web by 10-nm intermediate filaments, the tonofilaments. The membranes of adjacent cells do not fuse but are held apart by the material of the cell coat. At the center of this space is a thin dark line, the intermediate line, that may have cementing properties. Single half desmosomes are seen in some locations where epithelial cells abut on the basement membrane overlying the connective tissue (Fig. C58).

Figure C56. The desmosomes (D) in this electron micrograph of a section of the skin of a newborn rat appear as black structures between adjacent cells. Each desmosome consists of two hemidesmosomes, one from each of the adjacent cells. 12,500× Inset: 124,00×.

Figure C57. Electron micrograph of desmosomes in the stratified squamous epithelium of the cheek of a hamster. Each half desmosome is anchored to its cell web by 10-nm intermediate filaments, the tonofilaments. The membranes of adjacent cells do not fuse but are held apart by material of the cell coat. At the center of this space is a dark line, the intermediate line, that may have cementing properties. 70,000×.

Figure C58. In locations where a hemidesmosome's attentions are unrequited, such as the junction of an epithelial cell with its basement membrane, hemidesmosomes appear alone. In this electron micrograph of the base of epidermis of an amphibian, tonofilaments converge on the hemidesmosomes, which appear firmly affixed to the basement membrane. The material at the lower left is the connective tissue layer of the skin.

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Male Reproduction

Juho-Antti Mäkelä , Jorma Toppari , in Encyclopedia of Reproduction (Second Edition), 2018

Abstract

Intercellular bridges (ICB) are formed at the end of every cell division. In somatic cells they are transient structures and are quickly abscised resulting in formation of two daughter cells. In germ cells, however, the ICB structure is stabilized and the daughter cells are tethered together in a syncytium with a continuous cytoplasm. Synchronous differentiation of hundreds of spermatogenic cells within a syncytium is considered one of the outcomes of ICBs and the intercellular molecular traffic that they enable. Other hypothesized functions of ICBs include compensation of chromosome dosage in postmeiotic haploid cells, and sharing of essential signals and mRNAs within the syncytium. Stabilization of the ICBs is considered a differentiation commitment in the male germ-line and all spermatogenic cells develop in syncytia until the point of spermiation when mature elongated spermatids are disengaged from the seminiferous epithelium as single cells. Spermatogenic cell syncytia are stable, but they occasionally break and do not reach their theoretical maximal length. On the contrary, ICBs of spermatogonial stem cells (SSCs) are highly unstable and SSCs actively interconvert between single cell and short syncytial states. ICB are dynamic structures and they undergo changes in architecture, protein composition and diameter during the course of spermatogenesis. The fundamental importance of ICBs for male germ cell maturation is underlined by the fact that they are formed during spermatogenesis in diverse organisms from Drosophila to man, and lack of ICBs, as seen in TEX14 (testis-expressed gene 14) deficient male mice, results in azoospermia due to meiotic failure.

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Paracellular Channel Evolution

Jianghui Hou , in The Paracellular Channel, 2019

Abstract

Cell junction is a common feature of epithelial cells. During metazoan evolution, the form of cell junction has undergone considerable diversification. Epithelia in vertebrates develop tight junctions to control the diffusion of molecules through the paracellular space, whereas most invertebrates, such as Drosophila melanogaster and Caenorhabditis elegans, lack tight junctions but utilize septate junctions or hybrid junctions to establish the paracellular permeation barrier. Comparison among fly, worm, and mammal reveals marked conservation with respect to the proteins used for carrying out paracellular transport. Claudins and their evolutionally related proteins are vital to this process. Tricellular junction appears to be an exception. Invertebrate tricellular junction consists of a class of transmembrane proteins showing no homology to their vertebrate counterparts. Although vertebrate epithelia evolve to form tight junctions in place of septate junctions, two special types of vertebrate cells, the glial cells in the nervous system and the podocytes in the kidney, develop septum-like junctions bearing structural and functional similarities to invertebrate septate junctions, known as the paranodal junctions and the slit diaphragms respectively.

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Systems Cell Biology

K.A. Thomas , in Encyclopedia of Cell Biology, 2016

Destabilization of EC–EC Junctions

Intercellular junctions between ECs, mediated by adherens junctions and tight junctions, stabilize the vascular endothelial monolayer. These junctions need to be destabilized to allow sprouting ECs to migrate from existing microvessels in response to angiogenic inducers. ECs uniquely express the adherens junction transmembrane protein vascular endothelial-cadherin (VE-cadherin), which contains five extracellular Ig-like domains, a single-pass transmembrane helical sequence and C-terminal intracellular region. VE-cadherins from adjacent ECs are thought to form a zipper-like structure between them. Although the VE-cadherin crystal structure shows overlap between the N-terminal Ig-like domains on VE-cadherins from opposing directions, they are also thought to be able to generate narrower adherens junctions by overlapping multiple Ig-like domains as illustrated in Figure 7. VE-cadherin binds near the membrane to p120 that influences VE-cadherin retention and spatial organization at the cell surface. The C-terminal tail of VE-cadherin binds β-catenin that in turn binds α-catenin, which attaches to actin thereby linking cell surface intercellular junctions to intracellular cytoskeleton microfilaments (Bravi et al., 2014).

Figure 7. Endothelial junction structure and destabilization. Two types of endothelial cell junctions have been identified, both of which link transmembrane intercellular adhesion proteins to the intracellular actin cytoskeleton. Mutual binding of the N-terminal Ig-like domains of VE-cadherin molecules (blue) from adjacent ECs can create an adherens junction (top) and greater overlap among the Ig-like domains might be able to stabilize narrower junctions (center). The VE-cadherin intracellular domain binds p120 (green) and β-catenin (red), which is linked to actin (yellow) by α-catenin (purple). Narrower tight junctions (bottom) are formed by occludin (dark blue) and claudin (orange), two types of integral membrane proteins, each of which probably multimerizes and forms long ribbons that prevent diffusion of all but small molecules between ECs. VEGFR-activated signaling pathways can induce phosphorylation of VE-cadherin, β-catenin, occludin, and claudin destabilizing both adherens and tight junctions to induce vascular permeability and mobility.

EC tight junctions, also illustrated in Figure 7, are interspersed with adherens junctions but are thought to be more prevalent near the apical (luminal) surface. They limit passage of molecules through the EC–EC contact interface to approximately 800   Da. Tight junctions are composed of linear strands consisting of multiple proteins including occludins, EC-specific claudin-5, junctional adhesion molecules (JAMs), and zona occludens-1 (ZO-1), which links it to the actin cytoskeleton. VE-cadherin/β-catenin, acting through the PI3K/Akt pathway, expels a claudin-5 transcriptional repressor from the nucleus, effectively upregulating claudin-5 transcription and facilitating EC tight junction formation (Bravi et al., 2014).

The stability of the intercellular junctions is controlled by phosphorylation. VEGFR2, acting through endothelial nitric oxide synthase (eNOS) generation of nitric oxide, can nitrosylate β-catenin promoting its dissociation from VE-cadherin. VEGFR2-activated signaling pathways also phosphorylate VE-cadherin tyrosines in the binding sites for p120 and β-catenin, inhibiting adherens junction complex formation and promoting the internalization and degradation of VE-cadherin. Either the same or a different pool of β-catenin migrates to the nucleus and interacts with additional transcription factors to downregulate expression of the tight junction protein claudin-5. VEGF also induces serine phosphorylation of occludin, which increases ubiquitination that marks it for proteasome degradation. In addition to destabilizing EC intercellular junctions, growth factor-induced Rho GTPase activity promotes actin cytoskeleton contraction-mediated opening of the gaps between ECs. In contrast, Ang1-activated Tie2 signaling can inhibit VEGF-mediated EC junction destabilization, a process that can be functionally inhibited by the Tie2 weak agonist Ang2 (Goddard and Iruela-Arispe, 2013; Bravi et al., 2014). Therefore, as denoted in Figure 7, VEGF-induced destabilization of both adherens and tight junctions not only increases vascular permeability but also inhibits intercellular adhesion facilitating EC migration.

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Virus Assembly and Exit Pathways

Nicolas Cifuentes-Munoz , ... Rebecca Ellis Dutch , in Advances in Virus Research, 2020

2.4 Spread at cell junctions

Cell junctions are structures composed by several different transmembrane proteins, whose main function is to form a seal between polarized epithelial cells. Adhesion between epithelial cells is achieved by three main types of seals: tight junctions (TJ), adherens junctions (AJ) and desmosomes. These barriers cannot be penetrated by viruses unless there is substantial damage to the epithelia ( Mateo et al., 2015). However, epithelial cells can be infected from the apical or basolateral sides, with viral particles then transmitted to adjacent cells through specialized sites located in the vicinity of cellular junctions that are at least partially inaccessible from the extracellular environment. For example, in addition to cell-free spread, hepatitis C virus (HCV) can spread from cell-to-cell in a neutralizing antibody-independent manner (Brimacombe et al., 2011; Timpe et al., 2008). Claudin 1 (CLDN1) and occludin (OCLN), components of tight junctions, are critical for cell-to-cell spread of HCV (Brimacombe et al., 2011; Timpe et al., 2008). In addition, the cellular receptor CD81, scavenger receptor BI (SR-BI), apolipoprotein E, and low density lipoprotein receptor (LDLR) were shown to have a role in the cell-to-cell, but not cell-free, spread mechanism of HCV (Brimacombe et al., 2011; Fan et al., 2017; Timpe et al., 2008). Direct transmission of assembled HCV particles was proposed to occur in cell-cell contacts across partially sealed cell junctions (Brimacombe et al., 2011).

Robust evidence shows the use of adherens junctions by herpesviruses to spread from cell-to-cell (Johnson and Baines, 2011; Johnson and Huber, 2002). Herpes simplex virus type 1 (HSV-1) particles have been shown to be preferentially sorted towards cell junctions rather than to the apical surface of polarized epithelial cells (Johnson et al., 2001). The transport of particles towards these sites is primarily directed by the glycoprotein complex gI/gE, with the cytosolic domain of gE having a critical role (Dingwell et al., 1994; Dingwell and Johnson, 1998; Farnsworth and Johnson, 2006). Indeed, mutant viruses lacking gI/gE grow poorly in monolayers of human fibroblasts, and form plaques of small size (Dingwell et al., 1994). This phenotype was corroborated in vivo, as the △gI/gE mutant viruses produced small, punctate lesions in the corneal epithelium of rabbits and mice (Dingwell et al., 1994). The cell-to-cell spread mediated by gI/gE was found to be independent of neutralizing antibodies, suggesting that it occurs at sites of cellular junctions that are not accessible to these molecules (Dingwell et al., 1994). Immunofluorescent staining showed that gI/gE colocalized with the adherens junction protein β-catenin, but not with ZO-1, a component of tight junctions (Dingwell and Johnson, 1998). In addition, nectin-1, a component of adherens junctions, has been reported as a receptor for the herpes simplex virus glycoprotein D (Sakisaka et al., 2001; Yoon and Spear, 2002; Zhang et al., 2011). In this context, one accepted model is that gI/gE complexes accumulate at trans-Golgi network (TGN) subdomains, from where nascent virions are sorted to basolateral cell junctions for cell-to-cell spread (Farnsworth and Johnson, 2006; Johnson and Baines, 2011). At these sites, release of particles and their subsequent entry into the adjacent cell occur almost simultaneously. However, the critical function of gI/gE complexes extends beyond the spread between epithelial cells, and their role, together with pUS9, in the anterograde transport (from cell body to axons) of HSV-1 particles has been extensively studied (Howard et al., 2014; Kratchmarov et al., 2013; McGraw and Friedman, 2009; Snyder et al., 2008). Remarkably, the complex gI/gE together with pUS9 promote anterograde transport of enveloped particles, nucleocapsids, and glycoproteins through kinesin-mediated vesicular transport using microtubules (Diwaker et al., 2020; Johnson and Baines, 2011; Kratchmarov et al., 2013). The role of the gI/gE complex and US9 for cell-to-cell spread has additionally been reported for other herpesviruses including pseudorabies and VZV (Cohen and Nguyen, 1997; Diwaker et al., 2020; Johnson and Huber, 2002; Lyman et al., 2007; Zsak et al., 1992).

The use of desmosomes for cell-to-cell spread has been studied less than spread at the other cell junctions described above. However, some reports describe an important role of keratin 1 in infection by lymphocytic choriomeningitis virus (LCMV) (Labudova et al., 2009) (Labudova et al., 2019). Keratin filaments are an important component of desmosomes, and the LCMV nucleoprotein was shown to bind and stabilize keratin 1 (Labudova et al., 2009). This interaction resulted in increased desmosome formation and cell-cell adhesion capacity, facilitating cell-to-cell spread of LCMV (Labudova et al., 2009).

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New Insights into the Dynamics of Cell Adhesions

Patricia Costa , Maddy Parsons , in International Review of Cell and Molecular Biology, 2010

3.3 Proteins involved in cell–cell adhesion

Cell junctions are specialized cell–cell or cell–ECM contacts. In epithelial cells, cell–cell junctions are typically formed by AJ, and apical TJ. Desmosomes and GAP junctions are also found in certain groups of specialized epithelial cells. At the contacts between the cell and the ECM there is also formation of FAs and hemidesmosomes. Both TJs and desmosomes are formed by transmembrane adhesion proteins of the cadherin family whereas FAs and hemidesmosomes are formed predominantly by transmembrane proteins of the integrin family. Although endothelial cells show more flexible organized junctions they are able to form AJs and TJs as in epithelial cells ( Dejana, 2004). Tight junctions appear in epithelial cells at the most apical part of the cell. These junctions are composed of the transmembrane proteins claudin and occludin, and are associated with the ZO family of proteins, which can in turn bind to actin to form a mechanical scaffold (Fig. 2.1C). Epithelial AJs are composed of cadherin adhesion molecules. These epithelial cadherins are Ca2+-dependent transmembrane adhesion proteins that form homodimers at the plasma membrane between adjacent cells similar to a zipper-like structure (Cavey and Lecuit, 2009; Fukata and Kaibuchi, 2001).

At their cytoplasmic domain cadherins are known to bind actin through association with α-, β-, and p-120 catenins. Interaction of cadherins with the cortical actin cytoskeleton is a dynamic and tightly regulated process. This was traditionally thought to be due to direct physical interactions between E-cadherin and β-catenin, and indirectly to α-catenin thus connecting to actin filaments. However, recent biochemical and dynamics analysis have shown that this link may not exist and that instead, a constant shuttling of α-catenin between cadherin/β-catenin complexes and actin may be key to explain the dynamic aspect of cell–cell adhesion (Drees et al., 2005; Yamada et al., 2005). After formation of cell–cell contact that is dependent upon actin polymerization, formation of membrane protrusions and ruffles, cadherin homodimers start to cluster at contacting sites (Kametani and Takeichi, 2007). Clustering of cadherins leads to generation of acto-myosin tension at contact sites, which in turn generates a "pulling" force that facilitates formation of thick actin bundles resulting in contact expansion (Cavey and Lecuit, 2009) (Figure 2.2A).

Desmosomes are specialized cell–cell junction sites that can bind intermediate filaments (e.g., keratin, desmin) at their cytoplasmic domain (Figure 1.1C). Desmosomes are composed of three major gene families: cadherins (such as desmogleins and desmocollins), armadillo proteins (such as plakoglobin, plakophilins), and plakins (such as desmoplakin) the latter of which link to the intermediate filaments. Lateral interactions among proteins in the junctional plaque reinforce its stability (Green and Simpson, 2007). Following contact, desmosomal proteins are recruited to sites of cell–cell apposition in two phases; one fast from local pools of protein, and one slower recruitment phase from translocating particles in the cortical region of the cell (Godsel et al., 2005). The membrane-associated components of the adhesion are associated with the microtubule network, whereas desmoplakin associates with intermediate filaments. These relative associations play a role in strengthening the adhesive plaque over time. Once formed, desmosomes in intact epithelial layers are relatively immobile. However, studies have shown that these structures become considerably more dynamic upon insult, such as wounding of the skin (Green and Simpson, 2007). This is often coupled to increased levels of active PKC that may lead to phosphorylation of one or more of the components of desmosomal adhesive plaques thus modulating dynamic changes (Wallis et al., 2000).

GAP junctions are specialized cell–cell junctions in which the plasma membrane of the adjacent cells is penetrated by protein assemblies called connexons (Kojima et al., 2007) (Figure 2.1C). The apposed lipid bilayers are penetrated by connexons, each of which is formed by six connexin subunits. Two connexons join across the intercellular gap to form a continuous aqueous channel connecting the two cells. Gap junctions are composed of clusters of connexons that allow molecules smaller than about 1000 daltons to pass directly from the inside of one cell to the inside of the next. Cells connected by gap junctions share many of their inorganic ions and other small molecules and are therefore chemically and electrically coupled (Kjenseth et al., 2010). Gap junctions are important in coordinating the activities of electrically active cells, and they have a coordinating role in other groups of cells as well. While the dynamics of molecule transport through GAP junctions have been well described, dynamic changes in the proteins within the junctions themselves remain poorly understood.

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Visual System

Lee Ann Remington OD, MS, FAAO , in Clinical Anatomy and Physiology of the Visual System (Third Edition), 2012

Intercellular Junctions

Intercellular junctions join epithelial cells to one another and to adjacent tissue; some are named by their type and some by their shape. Protein components of intercellular junctions include cell adhesion molecules, transmembrane proteins (occludin, claudin), junctional adhesion molecules, and associated cytoplasmic proteins. 7 Junctions between cells or with connective tissue can have additional functions other than adhesion. Physical changes, such as pressure and biochemical or pharmaceutical factors, can modulate junctions and alter the junctional proteins. This allows information about changes in the extracellular environment to be relayed to the cell interior affecting intracellular processes.

In a tight (occluding) junction, the outer leaflet of the cell membrane of one cell comes into direct contact with its neighbor. Ridgelike elevations on the surface of the cell membrance fuse with complementary ridges on the surface of a neighboring cell. 8 As the paired strands meet, the neighboring cell membranes are fused. 9 The fibers of tight junctions are connected to the cytoskeleton within the cell.

A tight junction that forms a zone or belt around the entire cell, joining it with each of the adjacent cells is called a zonula occludens (ZO) (Figure 1-5). In these zones, row on row of interwining ridges effectively occlude the intercellular space. A substance cannot pass through a sheet of epithelium whose cells are joined by ZO by passing between the cells, but must pass through the cell.In stratified epithelia, whose surface layer is constantly being sloughed and replaced from below, ZO, if present, will be located in the surface layer. The components of the tight junction are found in increasing numbers as a cell moves from its origin in the basal layer until, finally when the cell reaches the surface, its occluding junction is complete. 10 The complex formed by the junctional proteins in the ZO can be affected in some diseases, causing in a breakdown in the barrier function, allowing a pathway to open through the network. Currently, researchers are developing pharmaceuticals that will cause a temporary disruption of the barrier, and that would allow other drugs or substances to pass through the intercellular route. In some instances, ridges in a tight junction are fewer and discontinuous, resulting in a "leaky epithelium." 8

A zonula adherens (ZA), an intermediate junction, is a similarly-shaped zone. However, the adjacent plasma membranes are separated, leaving a narrow intercellular space that contains a glycoporotein material causing cell adhesion but allowing intercellular passage. 12 ZA junctions produce relatively firm adhesions. Adjacent to the adhering junction are fine microfilaments that extend from a plaque just inside the membrane to filaments of the cytoskeleton, contributing to cell stability. 8 A terminal bar consists of a zonula occludens and a zonula adherens side by side, with the tight junction lying nearest the cell apex. 1,8

Round, buttonlike intercellular junctions are either macula occludens (MO) or macula adherens (MA), depending on the type of adhesion.

A desmosome is a strong, spotlike attachment between cells (see Figure 1-5). A dense disc or plaque is present within the cytoplasm adajcent to the plasma membrane at the site of the adherence. Hairpin loops of cytoplasmic filaments called tonofilaments extend from the disc into the cytoplasm and link to keratin filaments in the cytoskelton, contributing to cell stability. Other filaments, transmembrane linkers, cadherins, extend from the plaque across the intercellular space, holding the cell membranes together and forming a strong bond. 12 The intercellular space contains an acid-rich mucoprotein that acts as a strong adhesive. 8

A hemidesmosome provides a strong connection between the cell and its basement membrane and underlying connective tissue. It contains similar intracellular components; the protein complex extends through the cell membrane to attach to keratin in the basement membrane. Bundles of filaments join the intracellular plaque to underlying connective tissue matrix, often attaching to a plaque embedded in the connective tissue. 10

Gap junctions are formed by a group of (usually six)proteins, called connexins, that span the cell membrane and unite with connexins of a neighboring cell, forming a channel called a connexon (see Figure 5-1). 13 These narrow channels allow rapid cell-to-cell communication, i.e., passage of small molecules and ions from one cell to another. A group of cells with such connection act like a syncytium, that is, a single cell with multiple nuclei.

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Human Gene Discovery for Understanding Development of the Inner Ear and Hearing Loss

Yoni Bhonker , ... Karen B. Avraham , in Development of Auditory and Vestibular Systems, 2014

6 Junctions Between Cells: Gap and Tight Junctions

Cell junctions allow multicellular organisms to form tissue compartments with distinct ionic compositions by limiting the permeability of their constituents across epithelial barriers. In the inner ear, tight junctions are responsible for maintaining the different chemical compositions of the endolymph and perilymph ( Kitajiri et al., 2004), while gap junctions are involved in ionic and metabolic coupling of sensory epithelial cells (Qu et al., 2012). Both types of junctions are required for hearing, as mutations in junction proteins result in deafness.

Connexin 26 (Cx26), a gap junction protein encoded by the gene GJB2, was the first gene shown to cause non-syndromic HL DFNB1 (Kelsell et al., 1997) and is among the most prevalent genes in hereditary HL (Hilgert et al., 2009). Over 100 mutations have been found in this relatively small gene (Connexin-Deafness Homepage, http://davinci.crg.es/deafness/, accessed 14 Jan 2014). Gap junctions are created when six connexin subunits form a hexamer hemi-channel and two such hemi-channels, each located in a different cell, bind to each other and essentially couple the cells' cytosols both electrically and chemically. In the cochlea, gap junctions are comprised primarily of connexin 26 and connexin 30 (Cx30; encoded by GJB6) (Forge et al., 2003). However, only connexin 26 is absolutely required for hearing: when Cx26 was over-expressed in Cx30-null mice, normal hearing was restored, suggesting that Cx26 can form homomeric channels that functionally compensate for the absence of Cx30 (Ahmad et al., 2007); a complementary experiment in which Cx30 was over-expressed in Cx26-null mice did not restore normal hearing (Qu et al., 2012). While mutations in Cx30 have been reported to cause HL in humans (Grifa et al., 1999), it seems that this is primarily by reducing expression of Cx26 (Ortolano et al., 2008). It is worth noting these genes share at least some of their genetic regulatory elements.

Cx26 has a developmental role in the cochlea, as a targeted deletion of Gjb2 causes post-natal developmental arrest of the organ of Corti (Wang et al., 2009). The prevailing theory regarding gap junctions' role in hearing is their mediation of potassium recycling in the organ of Corti, following influx of the potassium-rich endolymph into the hair cells (Gerido and White, 2004). A competing theory suggests that gap junctions are responsible for transporting metabolites, specifically glucose, to the avascular sensory epithelium, and this process leads to a reduction in mitochondrial ATP production and hence to an increase in reactive oxygen species that ultimately cause cell death (Chang et al., 2008).

Tricellulin is encoded by the TRIC gene, responsible for DFNB49 non-syndromic HL (Riazuddin et al., 2006b). Tricellulin localizes to tricellular and bicellular tight junctions, where it is predicted to control macromolecule movement and ion permeability (Krug et al., 2009). Tight junctions in the inner ear are responsible for maintaining the different chemical compositions of the endolymph and perilymph.

A knock-in mouse model was generated with the corresponding human mutation to investigate the role of TRIC in the ear (Higashi et al., 2013; Nayak et al., 2013). These Tric R497X/R497X mice showed early-onset progressive HL. Microscopic examination and molecular analysis revealed that while tricellulin was expressed, it was unable to localize to tricellular tight junctions and that this was the only defect in cytoskeleton formation. The loss of tricellulin may affect the chemical composition of the endolymph, which may become toxic to hair cells as a result of these changes. To test this hypothesis, Tric R497X/R497X mice were crossed with Pou3f4 delJ mice (described above and in Minowa et al., 1999), predicting that the severe changes to the endolymph would prevent the formation of the toxic environment, possibly by removing the extracellular factor damaging the hair cells. Their results supported this hypothesis.

Furthermore, the availability of a mouse model designed specifically to resemble one of the human mutations allowed the researchers to conduct a broad phenotypic analysis on the mice. Additional abnormalities were found, suggesting that mutations in TRIC in humans may also have more phenotypic consequences beyond HL and that the patients should be examined more carefully.

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